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      In Vivo Phosphorylation of Ser 21 and Ser 83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase*

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          Abstract

          Background: Activation of yeast trehalase has been a convenient read-out for nutrient signaling to PKA, but demonstration of phosphorylation in vivo is lacking.

          Results: Nutrient activation is associated with phosphorylation, but phosphorylation is not enough for activation.

          Conclusion: Nutrient activation of trehalase is a reliable read-out for nutrient activation of PKA in vivo.

          Significance: Nutrient-sensing mechanisms can be identified using trehalase activation as a read-out.

          Abstract

          The readdition of an essential nutrient to starved, fermenting cells of the yeast Saccharomyces cerevisiae triggers rapid activation of the protein kinase A (PKA) pathway. Trehalase is activated 5–10-fold within minutes and has been used as a convenient reporter for rapid activation of PKA in vivo. Although trehalase can be phosphorylated and activated by PKA in vitro, demonstration of phosphorylation during nutrient activation in vivo has been lacking. We now show, using phosphospecific antibodies, that glucose and nitrogen activation of trehalase in vivo is associated with phosphorylation of Ser 21 and Ser 83. Unexpectedly, mutants with reduced PKA activity show constitutive phosphorylation despite reduced trehalase activation. The same phenotype was observed upon deletion of the catalytic subunits of yeast protein phosphatase 2A, suggesting that lower PKA activity causes reduced trehalase dephosphorylation. Hence, phosphorylation of trehalase in vivo is not sufficient for activation. Deletion of the inhibitor Dcs1 causes constitutive trehalase activation and phosphorylation. It also enhances binding of trehalase to the 14-3-3 proteins Bmh1 and Bmh2, suggesting that Dcs1 inhibits by preventing 14-3-3 binding. Deletion of Bmh1 and Bmh2 eliminates both trehalase activation and phosphorylation. Our results reveal that trehalase activation in vivo is associated with phosphorylation of typical PKA sites and thus establish the enzyme as a reliable read-out for nutrient activation of PKA in vivo.

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          Most cited references54

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          A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae.

          Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.
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            Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

            The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.
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              Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast.

              The two-hybrid system is a powerful technique for detecting protein-protein interactions that utilizes the well-developed molecular genetics of the yeast Saccharomyces cerevisiae. However, the full potential of this technique has not been realized due to limitations imposed by the components available for use in the system. These limitations include unwieldy plasmid vectors, incomplete or poorly designed two-hybrid libraries, and host strains that result in the selection of large numbers of false positives. We have used a novel multienzyme approach to generate a set of highly representative genomic libraries from S. cerevisiae. In addition, a unique host strain was created that contains three easily assayed reporter genes, each under the control of a different inducible promoter. This host strain is extremely sensitive to weak interactions and eliminates nearly all false positives using simple plate assays. Improved vectors were also constructed that simplify the construction of the gene fusions necessary for the two-hybrid system. Our analysis indicates that the libraries and host strain provide significant improvements in both the number of interacting clones identified and the efficiency of two-hybrid selections.
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                Author and article information

                Journal
                J Biol Chem
                J. Biol. Chem
                jbc
                jbc
                JBC
                The Journal of Biological Chemistry
                American Society for Biochemistry and Molecular Biology (9650 Rockville Pike, Bethesda, MD 20814, U.S.A. )
                0021-9258
                1083-351X
                28 December 2012
                15 November 2012
                15 November 2012
                : 287
                : 53
                : 44130-44142
                Affiliations
                From the []Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium,
                the [§ ]Department of Molecular Microbiology, VIB, Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Flanders, Belgium, and
                the []Netherlands Proteomics Center, Biotechnology Department, Delft University of Technology, 2628BC Delft, The Netherlands
                Author notes
                [1 ] To whom correspondence should be addressed: Kasteelpark Arenberg 31, B-3001 Leuven-Heverlee, Belgium. Tel.: 32-16-321507; Fax: 32-16-321979; E-mail: johan.thevelein@ 123456mmbio.vib-kuleuven.be .
                Article
                M112.421503
                10.1074/jbc.M112.421503
                3531729
                23155055
                22546c16-c7e1-4ca9-8d2b-22c6917f5051
                © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

                Author's Choice—Final version full access.

                Creative Commons Attribution Non-Commercial License applies to Author Choice Articles

                History
                : 20 September 2012
                : 30 October 2012
                Categories
                Signal Transduction

                Biochemistry
                enzyme mechanisms,phosphorylation,protein kinase a (pka),signaling,yeast metabolism,14-3-3,nutrient activation,nutrient signaling,protein kinase read-out,trehalase

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